Genital herpes simplex virus type 2 (HSV-2) is a sexually transmitted infection that disproportionately affects women. HSV-2 seroprevalence is nearly two-fold higher in women than in men in the US, and worldwide, nearly 100 million more women than men are HSV-2 infected. In sub-Saharan Africa, HSV-2 infection is one of the main drivers of the HIV epidemic. While a vaccine against HSV-2 would greatly benefit women's health, attempts at vaccine development have failed thus far. Despite success in animal models and induction of potent immune responses in humans, the glycoprotein based vaccines do not protect against HSV-2 acquisition. Recent small studies have described multiple-strain HSV-2 infection but the prevalence and risk factors for this are not known. Importantly, if multiple-stran infection is common, it will suggest that vaccines will need to produce immune responses that are more potent than those produced in natural infection. Our goal is to define whether, and to what extent, multiple-strain HSV-2 infection occurs, as that will guide whether new vaccines need to elicit better than nature immunity. Our Specific Aim is to determine the gender- specific prevalence of and risk factors for multiple-strain HSV-2 infection. We hypothesize that 1) women will be more likely than men to have genital HSV-2 infection with multiple strains; 2) persons with higher numbers of lifetime sex partner will be more likely to have genital HSV-2 that those with fewer partners; 3) Women from sub-Saharan Africa will have higher prevalence of multiple strain infection as compared with U.S. women; 4) HIV seropositive persons will have a higher prevalence of multiple strain infection as compared with HIV seronegative persons. We have prospectively collected unique sets of serial HSV-2 DNA positive genital samples from >800 participants enrolled in our extensive virologic and epidemiologic studies. These include samples from women and men from the US and sub-Saharan Africa, and men from Peru. HIV seropositive persons are included both in the US and African samples. All participants have clinically and virologically characterized genital HSV-2 infection, and provided detailed sexual histories. Access to these specimens places our group in a unique position to address these hypotheses. Our technical approach is to use Next-Generation Sequencing (NGS) to identify hypervariable regions and prevalent SNPs in 48 genital swab samples, and then use identified regions to perform high-throughput, multi-locus genotyping using pyrosequencing on specimen sets from 600 participants, to accurately characterize each within-person specimen set as single vs. multiple strain infection. Together, these data will clarify the relationship between gender an HSV-2 susceptibility and provide a benchmark for the likely efficacy of HSV-2 vaccines.